Biomarker detection device

ABSTRACT

The disclosure is directed to a device that includes an upper container configured to receive a fluid sample collected from a mammal into a first opening, the first opening opposite a second opening and a membrane covering at least a portion of the second opening, the membrane configured to allow transmission of a portion of the fluid sample through the membrane.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/442,235 filed on Jan. 4, 2017, the entire contents of which isincorporated herein by reference.

FIELD OF THE DISCLOSURE

The disclosure relates generally to the field of methods and devices fordetecting biomarkers. More specifically, the present disclosure isdirected to methods and devices that detect biomarkers in fluid samples.

BACKGROUND OF THE INVENTION

Biological fluid collection to identify and analyze different markers ofdiseases and symptoms occurs in health care settings. The fluidscollected are varied, including urine, blood and others.

Biomarkers, which are included in fluid samples, can be used for diseasedetection and monitoring. The biomarkers serve as indicators for thephysiological status at a given time and change, during the diseaseprocess. The search for disease-related biomarkers for early diseasedetection has been to look for proteins/metabolites/molecules that arealtered/secreted as a consequence of the disease process, and are shedinto body fluids. After collecting these body fluids, a laborious andtime-consuming process must be conducted to identify each candidatebiomarker(s) from among the thousands of intact and altered molecules inthe body fluids that are collected for analysis.

What is desired are methods and devices that can indicate the presenceof one or more biomarkers.

Embodiments of the present application provide devices and methods thataddress the above and other issues.

SUMMARY OF THE INVENTION

The disclosure is directed to a device that includes an upper containerconfigured to receive a fluid sample collected from a mammal into afirst opening, the first opening opposite a second opening and amembrane covering at least a portion of the second opening, the membraneconfigured to allow transmission of a portion of the fluid samplethrough the membrane.

This disclosure is also directed to methods of using the device. Thesemethods include providing a fluid sample to an upper container, theupper container configured to receive the fluid sample, which has beencollected from a mammal, into a first opening of the upper container,the first opening opposite a second opening of the upper container. Nextat least a portion of the fluid sample is transmitted through a membranecoveting at least a portion of the second opening. The portion of thefluid sample transmitted through the membrane can then enter a lowercontainer, with the lower container and upper container attachablyconnected to each other by a linking member, which is configured tomaintain the upper container vertically above the bottom container andis configured to maintain the membrane therein. The linking member canbe comprised of an upper portion and lower portion and can be configuredto maintain the membrane at any angle. The membrane can be any suitablemembrane for maintaining one or more antibodies, including strips suchas diagnostic strips.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are illustrations of the use of one embodiment of thedisclosed device.

FIG. 2 is an illustration of another embodiment of the disclosed device.

FIG. 3 is an illustration of a linking member of the disclosed device.

FIG. 4 is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 5 is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 6 is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 7 is an illustration of a membrane support.

FIG. 8 is an illustration of another embodiment of a membrane support.

FIG. 9 is an illustration of another embodiment of a membrane support.

FIG. 10 is an illustration of another embodiment of a membrane support.

FIG. 11 is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 12 is an illustration of a cross section of the linking member ofFIG. 11 .

FIG. 13 is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 14 is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 15 is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 16A is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 16B is an illustration of another embodiment of a linking member ofthe disclosed device.

FIG. 17 is an illustration of another embodiment of a linking member ofthe disclosed device.

FIGS. 18A-18D are photographs of an embodiment of a linking member andoperably attached containers.

FIGS. 19A and 19B are photographs of used testing strips.

FIG. 20 is a photograph of used testing strips within membrane supports.

FIGS. 21, 22, 23, 24, 25, 26 and 27 are lists of biomarkers that can bedetected with embodiments of the present disclosure.

DEFINITIONS

To facilitate the understanding of this invention a number of terms offin quotation marks in this Definitions section) are defined below. It isnoted that the drawings of the present application are provided forillustrative purposes only and, as such, the drawings are not drawn toscale. It is also noted that like and corresponding elements arereferred to by like reference numerals.

In the following description, numerous specific details are set forth,such as particular structures, components, materials, dimensions,processing steps and techniques, in order to provide an understanding ofthe various embodiments of the present application. However, it will beappreciated by one of ordinary skill in the art that various embodimentsof the present application may be practiced without these specificdetails. In other instances, well-known structures or processing stepshave not been described in detail in order to avoid obscuring thepresent application.

It will be understood that when an element as a layer, region orsubstrate is referred to as being “on” or “over” another element, it canbe directly on the other element or intervening elements may also bepresent. In contrast, when an element is referred to as being “directlyon” or “directly over” another element, there are no interveningelements present. It will also be understood that when element isreferred to as being “beneath” or “under” another element, it can bedirectly beneath or under the other element, or intervening elements aybe present. In contrast, when an element is referred to as being“directly beneath” or “directly under” another element, there are nointervening elements present.

As used herein, the term “substantially” or “substantial”, is equallyapplicable when used in a negative connotation to refer to the completeor near complete lack of an action, characteristic, property, state,structure, item, or result. For example, a surface that is“substantially” flat would either be completely at, or so nearly flatthat the effect would be the same as if it were completely flat.

As used herein, terms defined in the singular are intended to includethose terms defined in the plural and vice versa.

As used in this specification and its appended claims, terms such as“a”, “an” and “the” are not intended to refer to only a singular entity,but include the general class of which a specific example may be usedfor illustration, unless the context dictates otherwise. The terminologyherein is used to describe specific embodiments of the invention, buttheir usage does not delimit the invention, except as outlined in theclaims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weights, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersin the specification and claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and without limiting theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parametersdescribing the broad scope of the invention are approximations, thenumerical values in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains standarddeviations that necessarily result from the errors found in thenumerical value's testing measurements.

Thus, reference herein to any numerical range expressly includes eachnumerical value (including fractional numbers and whole numbers)encompassed by that range. To illustrate, reference herein to a range of“at least 50” or “at least about 50” includes whole numbers of 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers 50.1,50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In a furtherillustration, reference herein to a range of “less than 50” or “lessthan about 50” includes whole numbers 49, 48, 47, 46, 45, 44, 43, 42,41, 40, etc., and fractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4,49.3, 49.2, 49.1, 49.0, etc. In yet another illustration, referenceherein to a range of from “5 to 10” includes whole numbers of 5, 6, 7,8, 9, and 10, and fractional numbers 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, etc.

In the discussion and claims herein, the term “about” indicates that thevalue listed may be somewhat altered, as long as the alteration does notresult in nonconformance of the process or structure to the illustratedembodiment. For example, for some elements the term “about” can refer toa variation of ±0.1%, for other elements, the term “about” can refer toa variation of ±1% or ±10%, or any point therein.

As used herein, the term “biomarker” refers to a biomolecule, e.g., aprotein or a modified, cleaved or fragmented form thereof, a nucleicacid, carbohydrate, metabolite; intermediate, etc. which isdifferentially present in a sample and whose presence, absence orquantity is indicative of the status of the source of the sample (e.g.,cell(s), tissue(s), a mammal, etc).

As used herein, the term “antibody” means a population of immunoglobulinmolecules, which can be polyclonal or monoclonal and of any isotype. Asused herein, the term antibody encompasses an immunologically activefragment of an immunoglobulin molecule. Such an immunologically activefragment contains the heavy and light chain variable regions, which makeup the portion of the antibody molecule that specifically binds anantigen. For example, an immunologically active fragment of animmunoglobulin molecule known in the art as Fab, Fab′ or F(ab′)2 isincluded within the meaning of the term antibody.

DETAILED DESCRIPTION

Embodiments of the present disclosure relate to devices and methods ofdetecting one or more biomarkers. The methods and devices in thisdisclosure can be operated in a point of care testing scenario. Morespecifically, the methods and devices in this disclosure can be operatedat a site of patient care, such as a physician's office or a patient'shome, and can deliver results of biomarker presence in minutes to tensof minutes. The methods and devices in this disclosure can also avoidthe need for sending samples to diagnostic laboratories, which can takea long time to provide responsive results.

An overview of the disclosed devices and methods is provided below,before a more detailed discussion of the various embodiments.

The disclosure is generally directed to relatively quick biomarkerdetection of relatively low or trace quantities of biomarkers in afluid, with a device configured to capture the biomarkers and provide aprognosis/diagnosis of a disease/symptom at a collection site or someother suitable location. The use of the device has several advantagesagainst current collection/detection methods. The device is a one-stepsample collection and biomarker detection device that is used in manydifferent health care scenarios, where fluid samples (like urine forexample) are collected and sent to clinical labs for analysis.

The present device can be used as a point-of care device and caneliminate the lengthy process of transferring and analyzing the samplein specialized laboratory settings. The device can be used not only as asingle-use device, but can be modified to be a multiplex as well asrepeat-use device with the same sample that was collected for analysis.Thus, tests can be performed in clinics as well as by the patientsthemselves for diagnosing diseases and symptoms. Also, the presentdevice can be adopted for use in any analytical (such as testing forsubstances of abuse), pollution control, including detection ofenvironmental contaminants or other condition that relies on theidentification of relatively low concentration substances in volumes ofsolutions.

The first embodiment of the device is discussed below. The firstembodiment of the device for the detection of one or more biomarkers isshown in FIGS. 1A-1C. Although biomarkers can be detected by thedisclosed devices and methods, the devices and methods could be modifiedaccordingly to detect substances of abuse, analytes and markers insuitable liquids.

A device 1 for detecting one or more biomarkers includes a membrane 4and an upper container 8. Membrane 4 includes one or more firstantibodies 3, which are bound to the membrane in any suitable way, suchas by adsorption, absorption or otherwise being adhered to membrane 4,the one of more first antibodies being capable of binding to one or morebiomarkers 5 to form a complex. For illustration purposes the figures inthis application include illustrations of biomarkers and antibodies thatare quite larger than they appear in practice. These illustrations ofthe biomarkers and antibodies are not to scale and are for ease ofexplanation. The biomarker or biomarkers to be detected in the presentdevices and methods can be any suitable biomarker, including biomarkersthat are indicative of a specific disease state or physiological state.

In FIGS. 1A-1C, membrane 4 can be any material capable of antibodiesbeing bound thereto, capable of being in contact with fluids andconfigured to allow at least a portion of those fluids pass through themembrane into an optional lower container 7. The membrane can be one ormore of the following: cellulose based membrane such as a celluloseacetate, a regenerated cellulose membrane and/or nitrocellulose; vinylbased membrane such as polyvinylidene fluoride (PVDF); and carbon basedmembrane such as Perforene®, which is an about one atom thick graphenemembrane manufactured by Lockheed Martin®. In each of these embodiments,membrane 4 can include a plurality of openings to allow at least aportion of the fluids to pass through the membrane 4. These openings canbe of any suitable size that allows at least a portion of a fluid topass through and at least a portion of the biomarkers 5 to not passthrough. Some examples of the suitable sizes of these openings is about0.25 nm, about 0.5 nm, about 0.75 nm, about 1 nm, about 1.5 nm, about 2nm, about 2.5 nm, about 3 nm, about 3.5 nm, about 4 nm, about 4.5 nm,about 5 nm and greater. In some embodiments all openings of the membrane4 are the same size, in other embodiments the size of the openings ofthe membrane 4 vary across the membrane 4.

Upper container 8 is capable of holding a fluid 10, and can be made ofany suitable material, such as a plastic material. The fluid 10 can be abodily fluid that contains the one or more biomarkers to be detected.

The one or more antibodies 3 are selected based on the desired use ofdevice 1. Specifically, the one or more antibodies 3 can be selectedbased on the specific biomarker or biomarkers to be detected. As anexample, if the biomarker to be detected is Human Chorionic Gonadotropin(HCG), the antibody 3 would be one antibody, be bound to membrane 4 andcould be a monoclonal antibody against one epitope of HCG. In otherembodiments, if more biomarkers were to be detected, membrane 4 could becoated with one or more antibodies that are different from antibody 3.

Fluid 10 can be any bodily fluid that is capable of containing one ormore biomarkers 5 such as, but not exclusively, amniotic fluid, aqueoushumour, vitreous humour, bile, blood, breast milk, cerebrospinal fluid,cerumen, chyle, chyme, endolymph, perilymph, feces, gastric acid,gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid,pleural fluid, pus, rheum, saliva, sebum, semen, sputum, sweat, synovialfluid, tears, vaginal secretion, vomit, urine and combinations thereof.These fluids can be collected in any suitable way, such as with ahypodermic needle or collection upon excretion. These fluids can also becollected from any mammal, including a human.

Membrane 4 can be contacted with varying volumes of fluid 10, includingfrom about 1 μL to about 1 L of fluid 10. In another embodiment,membrane 4 can be contacted with about 1 mL to about 100 mL of firstfluid 10.

As fluid 10 contacts membrane 4, the one or more biomarkers 5 in fluid10 contact the one or more antibodies 3, the biomarkers and antibodiesbind and form a complex 11 with each other, as illustrated in FIG. 1B.To form the complex 11, the fluid 10 at least partially passes throughthe membrane 4 (so as to form passed through fluid 12, which can be anyportion of fluid 10), so that the fluid 10 at least partially contactsone or more antibodies 3.

As an example, of a fluid 10, there are more than 365 components innormal urine. Some of them are: Water (95%), Urea, Ions, Proteins,Sugar, Nitrite, Ketone, Bilirubin, Urobilinogen, Red and White BloodCells, Creatinine, Bacteria, Epithelial cells, etc. Size of componentsfound in urine are provided in Table 1.

TABLE 1 Na+ 0.102 nm (radius) K+ 0.138 nm (radius) Water Molecule 0.275nm (diameter) Cl− 0.181 nm (radius) Urea  0.26 nm (angstroms) Proteins 2-3 nm

A water molecule is around 0.275 nm in size and most proteins are above3 nm. The membrane 4 can be designed so that portions of the fluid (forexample water having a size of about 0.275 nm) passes through openingsof the membrane 4 and that elements such as biomarkers cannot passthrough openings of the membrane 4 (about 2-3 nm).

After first fluid 10 has contacted membrane 4, an optional second fluidcan contact the membrane 4, and at least a portion of that second fluidmay pass through membrane 4. The second fluid can contain one or moresecond antibodies 13 (as shown in FIG. 1C) that are capable of alsobinding to the one or more biomarkers 5 of the complex 11. Second fluidcan be any compatible physiological solution, such as but not limited tophosphate buffered saline and tris buffered saline, which is capable ofpreserving the structure and function of the one or more secondantibodies 13.

On to the one or more second antibodies 13 can optionally be one or morelabels. The one or more labels can be attached in any suitable way tosecond antibodies 13, such as being absorbed, being conjugated or linkedto second antibodies 13. After the second fluid contacts membrane 4, theone or more second antibodies 13 binds to the one or more biomarkers 5of complex 11. The one or more labels that are absorbed on to the one ormore second antibodies 13 are therefore attached to membrane 4. The oneor more labels can be any label that is suitable for absorption onto theone or more second antibodies 13 and that is able to change theappearance of the membrane 4, such as by a change in color of themembrane 4 in a region that is coated with the one or more antibodies 3.A non-limiting list of possible labels includes colloidal goldparticles, latex particles, single-walled carbon nanotubes (SWCNT),europium particles, terbium particles, samarium particles, dysprosiumparticles, Alexa Fluor® dyes, Qdots®, R-phycoerythrin (R-PE), biotin orany suitable fluorophore.

Prior to the second fluid contacting membrane 4, an optional third fluidcan contact membrane 4. This third fluid can contact membrane 4 and aidin removing any proteins that are nonspecifically contacting membrane 4.Nonspecific proteins include other biomarkers other than biomarkers thatare capable of binding with the one or more antibodies 3.

The method of using device 1 of the first embodiment is now described.

Initially, an amount of a fluid 10 is collected and is then placed incontact with membrane 4 in upper container 8, and at least a portion offluid 10 passes through membrane 4. The membrane 4 includes one or moreantibodies 3 that are capable of binding to the one or more biomarkers 5in fluid 10. Fluid 10 and membrane 4 can be in contact, as the fluid 10is passing through membrane 4, for any suitable amount of time, such as,for example, between about 1 second and about 10 minutes, or about 1minute and about 20 minutes, to create a complex 11 between the one ormore biomarkers 5 of the fluid 10 and the one or more antibodies 3 ofthe membrane 4.

After fluid 10 contacts membrane 4, the membrane 4 can then optionallybe contacted with a second fluid, which contains one or more secondantibodies 13. The one or more second antibodies 13 can bind to thecomplex 11 after contact with membrane 4. The one or more secondantibodies 13 can be absorbed to one or more labels, which can be usedto identify the binding of the one or more antibodies 3 and one or moresecond antibodies 13 to the one or more biomarkers 5. Second fluid andmembrane 4 can be in contact for any suitable amount of time, such as,for example, between about 1 second and about 10 minutes, or about 1minute and about 20 minutes.

As an optional step, prior to contact with the second fluid, membrane 4can be contacted with a third fluid that is capable of removingbiomarkers or other proteins that do not bind to the one or moreantibodies 3. Third fluid and membrane 4 can be in contact for anysuitable amount of time, such as, for example, between about 1 secondand about 10 minutes.

As another optional step, after removal of membrane 4 from the secondfluid, the amount of the one or more labels on membrane 4 can bequantified. For example, if the one or more labels were fluorescentlabels, membrane 4 could be quantified in a fluorescent intensity readerto determine the concentration of label or labels present.

Further embodiments of the device are discussed below in reference toFIGS. 2-10 .

In FIG. 2 , a device 20 is shown that includes an upper container 28 anda lower container 27, shown as being attached to one another through alinking member 21. The upper container has a first opening 28 a, whichmay or may not be sealed by a cover or the like, and a second opening 28b which can attach to a linking member 21. Fluid can be added to device20 through the first opening 28 a.

Each of upper container 28 and lower container 27 are attached to thelinking member 21 in any suitable way, such as a thread and grooveelement, a snap fit and/or an adhesive.

Within the linking member 21, a membrane support 22 can be housed. Thismembrane support 22 can be placed within linking member 21 in aconfiguration so that a membrane (not shown) can be exposed to a fluidin upper container 28 as the fluid at least partially passes through themembrane into the lower container 27. The membrane support 22 cart beremoved and replaced during a single use of the device 20, or removedand replaced for a subsequent use of the device 20.

A more detailed view of linking member 21 is shown in FIG. 3 , withoutthe containers attached and without the membrane support present. Inthis view a membrane support slot 23A is shown, which is substantiallyparallel with an upper surface 24 of the linking member 21A. Membranesupport slot 23A is so dimensioned as to attach to an upper containerthrough at least partial contact with upper container face 25 (withmembrane support slot 23A also being so dimensioned as to attach to anlower container through at least partial contact with a lower containerface (not shown)) and to accept a membrane support with little or noliquid exiting through any portion of the linking member 21 as the fluidpasses through linking member orifice 27.

FIG. 4 illustrates a modified linking member 21A, having a modifiedmembrane support slot 23A. Modified membrane support slot 23A includes aslot extension 26 to accept a portion of a membrane support.

FIG. 5 illustrates a second modified linking member 21B, having anangled membrane support slot 23B. Angled membrane support slot 23B isangled at about 10° from about parallel with the upper surface 24. Inother embodiments. Angled membrane support slot 23B can be angled atabout 5°, about 15°, about 20°, about 25°, about 30°, about 35°, about40° or about 45° from about parallel with the upper surface 24.

FIG. 6 illustrates a top view of second modified linking member 21B,having the membrane support 22 inserted therein. In this embodiment, amembrane 29 is visible through linking member orifice 27.

FIG. 7 illustrates a top view of one embodiment of membrane support 22,without a membrane, which includes a plurality of holes 30 in a membranesupport upper surface 32. Membrane support upper surface 32 isconfigured to support a membrane.

FIG. 8 illustrates a top view of an embodiment of a modified membranesupport 22A, which includes one opening in a center region of a modifiedmembrane support upper surface 32A. The modified membrane support uppersurface 32A is configured to support a membrane.

FIG. 9 illustrates a top view of an embodiment of the modified membranesupport 22A, with a modified membrane 29A supported by the modifiedmembrane support 22A.

FIG. 10 illustrates a top view of an embodiment of a second modifiedmembrane support 22B. The second modified membrane support 22B includesa second modified support upper surface 32B that includes openings tosupport the membrane 29.

A perspective view of the second modified linking member 21B (also shownin FIG. 6 ), having the membrane support 22 inserted therein is shown inFIG. 11 .

FIG. 12 illustrates a cross sectional view of second modified linkingmember 21B along line 12′ of FIG. 11 . In FIG. 12 , a fluid would pastupper container face 25, through linking member orifice 27, contactmembrane 29, at least a portion of the fluid passing through membrane29, and toward a lower container face 34 (which is so dimensioned as toattach to a lower container through at least partial contact with lowercontainer face 34) and a lower surface 36.

Another embodiment of a device 100 is shown in FIG. 13 . Device 100includes a linking member 121, which includes an upper portion 121 a anda lower portion 121 b. Although not shown, linking member 121 isdimensioned to operably attach to an upper container (not shown) bycontact with one or both of an upper surface 124 and an upper containerface 125. Although not shown, linking member 121 is dimensioned tooperably attach to a lower container (not shown) by contact with one orboth of a lower surface (not shown) and a lower container face (notshown).

Upper container face 125 includes an upper container face orifice 126that is configured to allow at least a portion of a fluid in an uppercontainer to pass therethrough. The diameter of the upper container faceorifice 126 can be adjusted for varying flow rates, for example, about0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm or more.

Membrane support 122 can include a membrane support orifice 128, whichis configured to allow at least a portion of a fluid that passes throughthe upper container face orifice 126 through the membrane supportorifice 128. Membrane support 122 is also configured to fit within anopen area created between the upper portion 121 a and the lower portion121 b.

The lower portion 121 b can include a lower portion upper face 136having a lower portion orifice 130 that is configured to allow at leasta portion of a fluid that passes through the membrane support orifice128 through the lower portion orifice 130.

Between upper container face orifice 126 and membrane support orifice128, membrane support 122 can include length of a strip 132 in a supportchannel. Similarly to membrane 29 discussed above, strip 132 can containone or more antibodies configured to bind with one or more biomarkers ina fluid. In other embodiments, strip 132 can be any diagnostic strip ordipstick.

The angle of the lower portion upper face 136 can be angled at about 5°,about 10°, about 15°, about 20°, about 25°, about 30°, about 35°, about40° or about 45° from about parallel with the upper container face 125.This angle of lower portion upper face 136 and the size of the uppercontainer face orifice 126 can be adjusted to arrive at a desired flowrate of fluid from upper container face orifice 126, through strip 132,to membrane support orifice 128 to lower portion orifice 130 and into abottom container. This flow rate can be any desired flow rate, such asabout 1 mL/minute, about 5 mL/min, about 10 mL/min, about 15 mL/min,about 20 mL/min, about 25 mL/min, about 30 mL/min, about 35 mL/min,about 40 ml/min, about 45 mL/min, about 50 mL/min or greater. Based onthe flow rate, the time for detection of biomarkers can be about 1minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15minutes, about 20 minutes or more.

Another embodiment of a device 101 is shown in FIG. 14 . Similarly todevice 100, device 101 includes a linking member 121 a, which includesan upper portion 121 ab and a lower portion 121 bb, but device 101 is amultiplex design, including three strips 134 a, 134 b and 134 c, whichcan each measure the same or different biomarkers.

In device 101, an upper container face 125 a includes three uppercontainer face orifices 126 a, 126 b, and 126 c and a membrane support122 a includes three strips 132 a, 132 b and 132 c each within a supportchannel 134 a, 134 b and 134 c, with each support channel including oneof a membrane support orifice 128 a, 128 b and 128 c.

A lower portion 121 b includes three lower portion orifices 130 a, 130 band 130 c. As an example, fluid enters upper container orifice 126 a,passes through strip 132 a, through membrane support orifice 128 a,through lower portion orifice 130 a and into a bottom container. Thefluid acts similarly for the b and c serialized elements in device 101.

The diameter of each of the upper container face orifices 126 a, 126 band 126 c can be adjusted for varying flow rates, for example, about 0.5mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm or more.

The angle of the lower portion upper face 136 a can be angled at about5°, about 10°, about 15°, about 20°, about 25°, about 30°, about 35°,about 40° or about 45° from about parallel with the upper container face125 a. This angle of lower portion upper face 136 a and the size of eachof the upper container face orifices 126 a, 126 b and 126 c can beadjusted to arrive at a same or different desired flow rate of fluidfrom upper container face 125 a to a lower container. This flow rate canbe any desired flow rate, such as about 1 mL/minute, about 5 mL/min,about 10 mL/min, about 15 mL/min, about 20 ml/min, about 25 ml/min orgreater. Based on the flow rate, the time for detection of biomarkerscan be about 1 minute, about 2 minutes, about 5 minutes, about 10minutes, about 15 minutes, about 20 minutes or more.

FIG. 15 illustrates a device 102, with the left image representing across section of device 102. Device 102 includes a linking member 121 b,which includes a lower portion 121 bc and an upper portion 121 ac.

Device 102 is similar to device 100, but device 102 includes a lowerportion upper face 136 aa that is about parallel with an upper containerface 125 aa. Also, the 121 bc includes a lower portion channel 138 thatcan receive a fluid from a membrane support orifice 128 ab, and transmitthat fluid to a lower portion orifice 130 aa. Also, in device 102 afterat least a portion of fluid from an upper container face 125 aa passesthrough a lower portion orifice 130 aa and into a lower container, amembrane support 122 b can be removed and replaced with a new membranesupport and the device 102 can be rotated about 180° so that the fluidin the lower container passes through a lower portion orifice 130 aa,the lower portion channel 138, a membrane support orifice 128 ab, andout an upper container orifice 126 aa into an upper container.

FIG. 16A illustrates a device 103, which is similar to device 100.Device 103 includes a linking member 121 c, which includes a lowerportion 121 bc and an upper portion 121 ad.

In device 103, the upper portion 121 ad includes an upper container face125 ab, which includes an upper face viewing portion 140. Viewingportion 140 can be see-through or substantially see-through so that astrip 132 is visible therethrough. In this embodiment, upper containerorifice 126 is further from the membrane support orifice 128 than theviewing portion 140. In other embodiments, the upper container orifice126 can be nearer the membrane support orifice 128 than the viewingportion 140, such as shown in FIG. 16B.

FIG. 17 illustrates a device 104. Device 104 includes a linking member121 d, which includes a lower portion 121 be and an upper portion 121ae. The upper portion 121 ae includes two upper container orifices 126ab and 126 ac, as well as two viewing portions 140 a and 140 b. Alsoincluded in device 104 is a membrane support 122 aa, having two supportchannels 134 aa and 134 ab, with corresponding membrane support orifices128 aa and 128 ab.

Therefore, the path of a fluid contacting an upper container face 125 acis, in a first path from upper container orifice 126 ab, through supportchannel 134 aa, membrane support orifice 128 aa and a lower portionorifice 130 ba, and in a second path from upper container orifice 126ac, support channel 134 ab, membrane support orifice 128 ab and a lowerportion orifice 130 bb.

As can be seen one upper container orifice 126 ab is further frommembrane support orifice 128 aa than the viewing portion 140 a, whileanother upper container orifice 126 ac is closer to membrane supportorifice 128 ab than the viewing portion 140 b.

Any element of any of the above described devices can be formed of anysuitable material. For example, elements can be formed of plasticmaterials, metal materials, naturally sourced materials, papermaterials, glass materials, rubber materials, carbon based materials andmixtures thereof.

The methods and devices of the present disclosure will be betterunderstood by reference to the following Examples, which are provided asexemplary of the disclosure and not by way of limitation.

Example 1

The presently disclosed device was tested for detecting trace biomarkers(considered to be less than about 1000 ng/mL concentration). Cotininedetection strips were placed in device 100. Cotinine is a metabolite ofnicotine and people who smoke cigarettes will eliminate cotinine throughurine (literature reports show that cotinine can accumulate in the bloodwithin 5 minutes of cigarette smoking and will be eliminated throughurine within 2-5 days). Device 100 was assembled with a cotininedetection strip (purchased from Craig Medical, Calif.) in the supportchannel (134 of FIG. 13 ).

Cotinine standard solution (400 ng/mL) was purchased from Sigma-Aldrich,Mo. Urine without cotinine and 400 ng/mL cotinine containing urine waspassed through the device. The results were observed after about 5minutes. The sample containing cotinine showed a positive band on thelateral flow cotinine strip.

Example 2

The following example was completed with a device 100 of FIG. 13 . 3Dprinting technology was used to create models, with a CubePro 3D printerbeing used to print the linking member and the membrane support out of,for example, Acrylonitrile butadiene styrene (ABS) material. Urinecollection cups were attached to the top and bottom of the linkingmember, as shown in FIG. 18A-18D.

In FIG. 18A, the upper container 28 is operably attached to the upperportion 121 a of linking member 121, and the lower container 27 isoperably attached to the lower portion 121 b. In FIG. 18B the upperportion 121 a is operably connected to the lower portion 121 b. In FIG.18C the membrane support 22 can be included, and can contact the linkingmember 121 as shown in FIG. 18D.

In FIG. 18D, the upper container face orifice 126 was 1 mm and a flowrate of fluid from the upper container 28 to the lower container 27 wasabout 50 ml, in about 5 minutes.

A pH detection strip 132 was added into the support channel 134.Solutions of normal (pH-7) and acidic (pH-2 by using HCl) were made andfirst the normal solution was poured into the upper container 128, thestrip 132 was replaced with an unused strip 132 and the acidic solutionwas poured into the upper container 28. Once each solution passed ontothe lower container 27, the membrane support was pulled out to see thechange in color of the pH strip 132. A clear and accurate differencebetween the two solutions was shown on the pH strips.

Example 3

The traditional method of urinalysis, by inserting the dipsticks intosimulated normal and high protein urine (purchased from CarolinaBiological Supply, NC) was performed, with the results shown in FIG. 19a . High albumin in sample is detected by the change in color on thedipstick.

Using the same set up as Example 2, albumin level was detected by aurinalysis strip 132 of membrane support 22. Normal and high proteinurine samples were analyzed by using the device (with results shown inFIG. 19 b ) and compared to the traditional analysis. The device 100derived results are similar to those achieved by routine analysis, withthe use of device 100 being more hygienic and sterile as compared totraditional urinalysis.

Example 4

The following example was completed with a device 103 b of FIG. 16 b.

Typical Human Chorionic Gonadotropin (HCG) biomarker detection devices(also known as home pregnancy tests) provide an indication of HCGpresence above about 250 mIU/mL. The ability of devices of the presentdisclosure was determined to detect biomarkers at levels lower thanabout 250 mIU/mL, for instance, about 25 mIU/mL.

Clarity HCG detection strip 132 was placed in membrane support channel134 in the linking member of FIG. 16B. Literature reports show thaturine HCG is less than 100 mIU/ml in the first 4 weeks of pregnancy andhence is difficult to detect positive HCG presence using typical testsduring this early time.

Normal urine was first passed through upper container face orifice 126,to contact strip 132. Strip 132, was then removed and replaced with anunused strip 132, and 25 miU/mL HCG containing urine was passed throughupper container face orifice 126 to contact the strip 132. The resultsshown in FIG. 20 were observed in 5 minutes after the urine was passedthrough. The sample containing 25 mIU/mL HCG created a positive band (onthe membrane support 122 numbered “2” in FIG. 20 ) the lateral flowstrip 132. A commercially available Clarity Diagnostics hCG Single StepUrine Cassette Pregnancy Test was also tested as instructed by ClarityDiagnostics. The commercially available test provided a positive resultfor urine of a concentration of 250 mIU/mL HCG, but did not provide apositive result for urine of a concentration of 25 mIU/mL HCG.

One reason the present device can be used to detect such low levels isthat is that every portion of the liquid to be tested (and allbiomarkers therein) flow through the detection strip that is present inthe membrane support, which allows for many markers to be confined tothe detection strip.

Example 5

Many different biomarkers can be detected with the presently discloseddevice. Below is a discussion of just some biomarkers that can bedetected.

Detection of urine based disease/symptom biomarker detection iscumbersome and difficult due to the collection methods as well as thetrace amounts of molecules that are to be detected. Over the years,biomarkers for diabetes associated kidney dysfunction, cancers,neurodegenerative diseases and even neuropsychiatric disorders have beenidentified and many of them are found to be in less than 1000 ng range.Some of these markers are shown in FIGS. 21-27 .

In FIG. 23 , urinary biomarkers of human neuropsychiatric disorders arelisted, with MDD being major depressive disorder, BD being bipolardisorder and ASD being autism spectrum disorder.

In FIG. 24 , urinary biomarkers of neurodegenerative diseases arelisted, with AD being Alzheimer's disease, PD being Parkinson's disease,MScl being multiple sclerosis and TSEs being transmissible spongiformencephalopathies.

FIG. 25 is a list of the relative abundance classes of urine proteinbiomarkers.

FIGS. 26 and 27 are lists of markers, with urine proteins identified byLC-MS/MS in type 2 diabetic patients with macroalbuminuria. Theidentified proteins are listed with their SwissProt or TrEMBL accessionnumbers. Molecular weight (MW) is calculated using the CalPI/MW tool.

The described embodiments and examples of the present disclosure areintended to be illustrative rather than restrictive, and are notintended to represent every embodiment or example of the presentdisclosure. While the fundamental novel features of the disclosure asapplied to various specific embodiments thereof have been shown,described and pointed out, it will also be understood that variousomissions, substitutions and changes in the form and details of thedevices illustrated and in their operation, may be made by those skilledin the art without departing from the spirit of the disclosure. Forexample, it is expressly intended that all combinations of thoseelements and/or method steps which perform substantially the samefunction in substantially the same way to achieve the same results arewithin the scope of the disclosure. Moreover, it should be recognizedthat structures and/or elements and/or method steps shown and/ordescribed in connection with any disclosed form or embodiment of thedisclosure may be incorporated in any other disclosed or described orsuggested form or embodiment as a general matter of design choice.Further, various modifications and variations can be made withoutdeparting from the spirit or scope of the disclosure as set forth in thefollowing claims both literally and in equivalents recognized in law.

What is claimed is:
 1. A device comprising: an upper container, theupper container configured to receive a fluid sample containing one ormore biomarkers collected from a mammal into a first opening, the firstopening opposite a second opening; a membrane, the membrane covering atleast a portion of the second opening, the membrane configured to allowtransmission of a portion of the fluid sample through the membrane; oneor more antibodies bound to the membrane, the one or more antibodiesconfigured to bind to the one or more biomarkers; a linking member, thelinking member comprising a linking member orifice, an upper surface, alower surface, a membrane support slot between the upper surface and thelower surface, and one or more of a thread and groove connection, a snapfit connection and an adhesive, wherein the one or more of the threadand groove connection, the snap fit connection and the adhesive areconfigured to maintain the upper container vertically above the linkingmember, and a membrane support, wherein the membrane support isdimensioned to fit within the membrane support slot, wherein themembrane support is configured to maintain the membrane within themembrane support slot, and wherein the membrane support is configured tobe removed from the membrane support slot.
 2. The device of claim 1,further comprising a lower container.
 3. The device of claim 2, whereinthe linking member comprises an upper portion and a lower portion. 4.The device of claim 3, wherein a membrane support of the linking memberis configured to maintain the membrane.
 5. The device of claim 3,wherein the membrane is maintained at an angle to an upper containerface of the upper portion.
 6. The device of claim 2, wherein themembrane is maintained within a membrane support of the linking member.7. The device of claim 6, wherein the membrane support comprises aplurality of holes in a membrane support upper surface.
 8. The device ofclaim 7, wherein the membrane support upper surface is configured tosupport the membrane.
 9. The device of claim 6, wherein the membranesupport comprises one opening in a center region of a membrane supportupper surface.
 10. The device of claim 9, wherein the membrane supportupper surface is configured to support the membrane.
 11. The device ofclaim 6, wherein the membrane support comprises a plurality of openingsin a membrane support upper surface.
 12. The device of claim 11, whereinthe membrane support upper surface is configured to support themembrane.
 13. The device of claim 2, wherein the membrane is maintainedat an angle to an upper container face of the upper portion.
 14. Thedevice of claim 2, wherein the linking member comprises an uppercontainer face that is configured to contact a fluid sample in the uppercontainer.
 15. The device of claim 2, wherein the linking membercomprises an upper container face, wherein each of the linking memberorifice and the upper container face extend over a portion of an upperportion of the linking member.
 16. The device of claim 1, wherein themembrane is selected from the group consisting of a cellulose basedmembrane, a vinyl based membrane and a carbon based membrane.
 17. Thedevice of claim 16, wherein the cellulose based membrane is selectedfrom the group consisting of a cellulose acetate, a regeneratedcellulose membrane and a nitrocellulose.
 18. The device of claim 16,wherein the vinyl based membrane is polyvinylidene fluoride (PVDF). 19.The device of claim 16, wherein the carbon based membrane is about a oneatom thick graphene membrane.
 20. The device of claim 1, wherein themembrane is a strip.
 21. The device of claim 1, wherein openings ofmembrane comprises openings of a size selected from the group consistingof about 0.25 nm, about 0.5 nm, about 0.75 nm, about 1 nm, about 1.5 nm,about 2 nm, about 2.5 nm, about 3 nm, about 3.5 nm, about 4 nm, about4.5 nm and about 5 nm.
 22. The device of claim 1, wherein the membraneis configured to transmit a portion of the fluid sample through themembrane as a lateral flow transmission.
 23. The device of claim 1,further comprising: one or more second antibodies configured to bind tothe one or more biomarkers; and one or more labels attached to the oneor more second antibodies.
 24. The device of claim 23, wherein the oneor more labels are configured to change a color of the membrane.
 25. Thedevice of claim 23, wherein the one or more labels are selected from thegroup consisting of colloidal gold particles, latex particles,single-walled carbon nanotubes (SWCNT), europium particles, terbiumparticles, samarium particles, dysprosium particles, R-phycoerythrin(R-PE), and biotin.
 26. The device of claim 1, wherein the membranesupport slot is at an angle of about 5° to about 45° from about parallelwith the second opening.